A difficulty in interpreting the significance of the well-documented deficiency of the alpha-ketoglutarate dehydrogenase complex in Alzheimer's Disease (AD) is that impaired energy/oxidative metabolism also occurs in other neurodegenerative disorders including the CAGn/Qn disorders. This project will test a number of mechanisms that might damage mitochondria, including KGDHC, in the CAGn/Qn disorders. We have shown that the enzyme tissue transglutaminase (tTGase; from guinea pig liver) can covalently attach Qn expansions to KGDHC and to glyceraldehyde-3-phosphate dehydrogenase, inactivating them. tTGase action of histone aggregates them. A variety of other proteins were not substrates. Mitochondrial damage might impair calcium regulation and thereby activate (calcium-dependent) transglutaminase, leading to a vicious spiral, even though transglutaminase and the aggregates which accumulate in the CAGn/Qn disorder are not found in mitochondria. Six series of experiments will be done. (I) The human brain transglutaminase(s), which appear to differ from the widely used guinea pig liver enzyme, will be purified, characterized, cloned, and sequenced. (ii) Since transglutaminase activity is almost absent from human fibroblasts, its activity will be increased by raising cytosolic Ca2+, by dexamethasone induction, and possibly by transfection. The presence and timing of abnormalities in the cells will be determined in relation to the increases in transglutaminase in terms of both time and quantity of enzyme induced. These studies will clarify whether damage to mitochondria (including to KGDHC) occurs in this model and whether it precedes or follows other evidence of cell damage. (iii) Proteins which are transglutaminase substrates in vitro (including KGDHC) will be examined in the brains of transgenic CAGn/Qn mice (with Project 5). These studies will determine whether changes in constituents of energy metabolism (including KGDHC) occur in these models and whether they precede or follow accumulation of aggregates. (iv) Rationally designed transglutaminase inhibitors will be tested with purified proteins, cultured cells, and in transgenic animals. (v) The utility of epsilon-(gamma- glutamyl)lysine as a marker for CAGn/Qn disorders will be tested, in human tissues as well as in animal and cultured cell models. If these studies indicate that mitochondrial damage in the CAGn/Qn disorders is a consequence of the accumulation of intracellular aggregates, future studies will focus on the effects of mitochondria (including KGDHC) of the aggregates that accumulate in AD as well as those in other neurodegeneration.